Identification and characterization of leukemia stem cells in
murine MLL-AF9 acute myeloid leukemia
Tim C.P. Somervaille1and Michael L. Cleary1,*
1Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Using a mouse model of human acute myeloid leukemia (AML) induced by the MLL-AF9 oncogene, we demonstrate that
colony-forming cells (CFCs) in the bone marrow and spleen of leukemic mice are also leukemia stem cells (LSCs). These
self-renewing cells (1) are frequent, accounting for 25%–30% of myeloid lineage cells at late-stage disease; (2) generate
a phenotypic, morphologic, and functional leukemia cell hierarchy; (3) express mature myeloid lineage-specific antigens;
and (4) exhibit altered microenvironmental interactions by comparison with the oncogene-immortalized CFCs that initiated
the disease. Therefore, the LSCs responsible for sustaining, expanding, and regenerating MLL-AF9 AML are downstream
myeloid lineage cells, which have acquired an aberrant Hox-associated self-renewal program as well as other biologic
features of hematopoietic stem cells.
Acute myeloid leukemia (AML) is a clonal neoplastic disorder
that originates from a single transformed cell, which has pro-
gressively acquired critical genetic or epigenetic changes that
disrupt key growth-regulatory pathways (Hanahan and Wein-
berg, 2000). Within the leukemia clone, there is significant cellu-
lar morphologic, phenotypic, and functional heterogeneity anal-
ogous to the hierarchical organization of normal hematopoiesis.
Notably, only a subfraction of cells are proposed to be leukemia
stem cells (LSCs) with the ability to self-renew extensively, and
to initiate, sustain, or regenerate the disease. Conversely, the
majority of cells are either transitional cells with limited prolifer-
ative capacity or more differentiated end cells (Mackillop et al.,
1983; Kummermehr, 2001). Evidence for a hierarchical cellular
organization of human AML derives from studies showing that
only a small proportion of AML cells are clonogenic in in vitro
culture (Buick et al., 1977), and that an even smaller fraction of
AML blood blasts, defined by a CD34+CD382surface pheno-
type, can transfer disease to immune-deficient mice (Lapidot
topoietic stem cells (HSCs) are also CD34+CD382, these and
other observations (Miyamoto et al., 2000; Hope et al., 2003)
have been taken to suggest that AML LSCs originate from and
reside exclusively within the most immature bone marrow
(BM) progenitor compartment. However, this lineal relationship,
which has important pathogenic and clinical implications, may
not always hold true. For example, a recent study of blastic
transformation of chronic myeloid leukemia proposed that cells
with a granulocyte-macrophage progenitor (GMP) cell pheno-
type were candidate LSCs (Jamieson et al., 2004).
is likely to be essential, and probably sufficient, for cure of dis-
ease, and thus increasing efforts have focused on attempting
to define the unique biological properties of AML LSCs by com-
parison with the transit and end cell compartments within the
leukemia, as well as with normal hematopoietic stem and pro-
genitor cells. However, an essential prerequisite for defining
these unique properties, so that targeted chemo- and immuno-
therapeutic strategies may be developed, is an accurate deter-
mination of the phenotype as well as the frequency of LSCs
within the AML clone. To date, such efforts have primarily
focused on xenografting human leukemia cells into immune-
deficient mice. A complementary, but currently unexploited,
approach is to characterize LSCs in mouse genetic models of
human leukemia, which have proven to be invaluable tools in
defining the mechanisms of disease pathogenesis. Two recent
sion of MLL-ENL or MOZ-TIF2 in progenitor cells with limited
self-renewal capacity, as well as in HSCs (Cozzio et al., 2003;
Huntly et al., 2004). However, these studies did not address
the critical issue of whether the respective murine leukemias
S I G N I F I C A N C E
An essential prerequisite for the development of more effective targeted therapies in AML is a characterization of the frequency and
biologicalproperties of LSCs. In a mouse modelof human MLL-AF9 AML, we have definedthe size and lineal derivation of the LSC com-
hypothesis that AML LSCs are always rare and solely located within the most immature bone marrow stem/progenitor compartment.
Furthermore, LSCs exhibit markedly different microenvironmental interactions, compared with cells simply immortalized by MLL-AF9,
indicating that acquisition of sensitivity to stromal cell-derived survival and proliferative signals is a critical feature of LSCs, in addition
to their extensive self-renewal capabilities.
A R T I C L E
CANCER CELL 10, 257–268, OCTOBER 2006 ª2006 ELSEVIER INC.DOI 10.1016/j.ccr.2006.08.020257
were sustained by a small fraction of self-renewing Lin2stem
and/or progenitor cells, as predicted by current models of AML
based on NOD/SCID transplantation assays, or alternatively,
whether the LSC compartment is larger and altogether distinct
from the normal stem and progenitor compartment.
We report here the identification and characterization of LSCs
in a mouse model of AML initiated by MLL-AF9, a frequently
occurring MLL fusion oncogene typically associated with the
FAB-M4 or M5 subtypes of human AML (Swansbury et al.,
1998).Our studiesindicate thatLSCs areneither rarenorsynon-
ymous with the stem and progenitor cells targeted by initiating
MLLmutations.Rather, LSCsin thismodel are frequent, located
almost exclusively downstream of the normal progenitor com-
partment by immunophenotype, and constitute myeloid lineage
cells that have acquired an aberrant self-renewal program as
well as other biologic features of HSCs, including substantially
altered microenvironmental interactions.
The leukemogenic potential of single MLL-AF9-immortalized
colony-forming cells (CFCs) was evaluated using a retroviral
transduction/transplantation assay that reproducibly reads out
the properties of MLL fusion oncogenes (Lavau et al., 1997).
BM stem and progenitor cells (c-kit+) from EGFP transgenic
mice were transduced with MLL-AF9 and serially replated in
semisolid medium (Figures 1A and 1B). At the end of the third
round of culture, individual colonies were isolated and ex-
panded further in either liquid or semisolid media (Figure 1A).
Fourteen lines (representative of ten unique clones) (Figure 1C)
were randomly selected and injected (106cells) into separate
sublethally irradiated, wild-type recipients. All mice developed
AML,with amedianlatencyof84.5 days(range60–121)(Figures
1Dand 1E;Tables S1 andS2 in the Supplemental Data available
with this article online). Southern blot analysis demonstrated
that each leukemia was clonally identical to its respective in-
jected cell population (Figure 1C). Therefore, immortalized
colony-forming cells (ICs) transformed by MLL-AF9 in vitro con-
sistently possess the potential to initiate leukemias in vivo, dem-
onstrating a high correlation of CFC activity with leukemogenic
potential in this model of AML.
LSCs are frequent in mice with MLL-AF9 myeloid
A similar approach was employed to analyze the leukemogenic
potential of CFCs derived from leukemic mice (Figure 2A).
Using semisolid culture assays, the CFC frequencies in the
BM and spleens of leukemic mice were 29.8% 6 4.1% and
Figure 1. MLL-AF9-immortalized CFCs have high
A: Schematic illustration of the experimental
approach employed to assess the correlation
of leukemogenic potential with CFC activity of
MLL-AF9-immortalized cells. BM stem and pro-
genitor cells (c-kit+) were transduced with MLL-
AF9 and then serially replated in methylcellulose
medium every 5 days, with G418 drug selection
in the first round. Single colonies (33 total over
five experiments) with either type I (16 each) or
type II (17 each) morphology (Lavau et al.,
1997) were plucked and individually expanded
in liquid (29) or semisolid (4) medium, and 106
cells were then transplanted into syngeneic re-
cipient mice. Single CFCs routinely expanded
to 106progeny cells within 12–15 days.
B: The mean (6SEM) number of colonies (R1000
cells) per 10,000 cells plated in each roundis indi-
cated. The clonogenic potential of progenitors
transduced with empty vector was exhausted
by the end of round two (data not shown). The
mean (6SEM) frequency of CFCs at the time of
transplant in the 14 lines derived from single
plucked colonies is indicated in the last column
C: Southern blot analysis (left panel) demon-
strates the integration sites in 14 separate trans-
planted lines derived from single immortalized
CFCs (neo probe of Stu1-digested genomic
DNA). A median of three integration sites per
sites (right panel) in three representative paired
transplanted lines (L) with their respective AML
cells (A) confirmed that they were clonally
D: Survival curve of animals transplanted with
cells (106) derived from single MLL-AF9-immortal-
E: Representative cytospin of splenocytes (May
Grunwald Giemsa stain) from a mouse with
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CANCER CELL OCTOBER 2006
support from the Children’s Health Initiative of the Packard Foundation and
PHS grants CA55029 and CA116601.
Received: March 14, 2006
Revised: July 27, 2006
Accepted: August 28, 2006
Published: October 16, 2006
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